Chaperones, which are known as classical folding helpers, are proteins that assist the folding and maintenance of the structural integrity of other proteins. They bind to denatured or hydrophobic surfaces of proteins and help in re-naturing and keeping proteins in solution. Due to their superior physico-chemical properties chaperones are used as folding assistants and fusion partners in protein technology. One class of chaperones is the family of FKBP chaperones, proteins that bind to the immunosuppressant drug FK506.
The use of FKBP chaperones like SlyD, FkpA and SlpA (=SlyD-like protein A) as fusion partners for difficult proteins has been widely described (WO 2003/000878, WO 2009/074318, EP 2127679).
Commercially available immunoassays for the detection of antibodies against pathogens like, e.g., human immunodeficiency virus (HIV), Rubella virus, cytomegalovirus (CMV) or herpes simplex virus (HSV) contain polypeptide fusion proteins wherein chaperones are fused to specific target antigen sequences. Such fusion proteins are described in, e.g., Scholz et al., J. Mol. Biol. (2005) 345, 1229-1242, Scholz et al., Biochemistry (2006) 45, 20-33 or Scholz et al., Biochemistry (2008) 47, 4276-4287.
SlyD, FkpA and SlpA possess outstanding solubilization (i.e. chaperone) properties and are characterized in that they are able to refold reversibly after chemically or thermally induced unfolding. As fusion partners for difficult target polypeptides they play at least a threefold role: firstly, they increase the production of target proteins that are heterologously overexpressed in procaryotic organisms, secondly, they facilitate and assist the in vitro refolding of the target polypeptides, and thirdly, they increase the overall solubility and stability of the respective target polypeptide.
However, chaperones like SlyD, FkpA and SlpA are immunogens in their own right. Since they are abundant bacterial proteins, they are recognized as non-self by the human (or, generally speaking, mammalian) immune system, triggering a powerful humoral immune response, which results in the production of specific antibodies with high affinity. A considerable percentage of adult human sera therefore contain significant immunoglobulin titers against these chaperones. As a consequence, there is a considerable likelihood that a human serum sample may turn out false positive in an immunoassay, in particular in an immunoassay of the double antigen sandwich format that uses antigen specifiers fused to bacterial chaperone modules.
In order to avoid such unwanted cross-reactions due to the antibody-induced bridging of fusion partners, immunoassays are usually designed in an asymmetric fashion. This means that for example in an immunoassay for the detection of antibodies designed in the well-known double antigen sandwich format (DAGS) a person skilled in the art uses different fusion partners for the applied antigens on both sides of the assay in order to avoid non-specific bridging. If identical fusion partners were used for the antigens on the solid phase and the detection side, interfering components in the sample could establish a bridge between said identical fusion partners and thus evoke a (false) positive reaction.
As a further means to prevent unwanted binding to a fusion partner which is part of an antigen-fusion protein, a chemically polymerized form of the employed fusion module (i.e. the fusion part without any specific antigen) is usually added to the assay in large excess. Due to their high epitope density and their high effective concentrations, these chemically polymerized fusion modules preferably allure, bind and quench those IgGs and IgMs that are directed towards said fusion module. The chemically polymerized fusion modules serve as a bait, and they quench the interfering compounds in the sample very efficiently so that interferences can be suppressed and ruled out. When, for instance, E. coli SlyD is used as a fusion partner for a given antigen in an immunoassay of the double antigen sandwich type, it would be highly advisable to generate chemically polymerized E. coli SlyD (by means of cros slinking with, e.g., glutardialdehyde) and to add this polymer to the assay as an anti-interference substance.
However, a considerable disadvantage in using chemically polymerized proteins lies in the chemical production process itself. Depending on the cross-linking agent applied, the chemical polymerization process is not entirely reproducible. The chemically cross-linked polymers usually show a large distribution of polymers of different size, i.e. they strongly vary with respect to connectivity and they are characterized by considerable heterogeneities. In order to select the effective polymer fractions (i.e. the polymer fractions with the desired anti-interference abilities) the polymer pool needs to be purified and fractionated by time-consuming and cumbersome chromatographic methods. In addition, only limited yields can be obtained as only a small percentage of the product will elute in the desired fraction.
In order to overcome the obstacle of using insufficiently characterized chemically polymerized material in immunoassays we searched for an alternative way to generate anti-interference substances. We strived to obtain anti-interference modules with a sufficiently high and well-defined epitope density in a simple and convenient manner. So we addressed the question whether it was possible to create a well-defined, highly soluble and highly efficient anti-interference module in an utterly recombinant fashion. Briefly, the problem to be solved was to obtain chaperone fusion partners in a soluble form, with high epitope density and in a reproducible and standardizable way.